One of the major challenges in molecular biology during this decade is the determination of three-dimensional structures of membrane proteins. The analysis of genomes from bacteria to humans reveals that as much as 30% of the total proteins are, indeed, membrane proteins. However, in spite of the fact that as much as 17,780 three- dimensional structures have been deposited in the PDB to date, only 0.56% of these structures are of membrane proteins. There are a number of reasons for this surprising lack of structural studies of membrane proteins. First, membrane proteins are highly hydrophobic, which makes it extremely difficult to purify them to the homogeneity required for structural studies. Second, the expression of membrane proteins is generally very poor. Is there a way to circumvent these problems? If one can produce only a single membrane protein of interest without producing any other cellular proteins in living cells, the structural study of membrane proteins can be carried out without purification. This will, no doubt, revolutionize the structural biology of membrane proteins and will make a major contribution to our understanding of the structure and function of membrane proteins. In this proposal, I will attempt to establish the technology needed to produce a single membrane protein of interest in living cells without producing any other cellular proteins. This technology, termed the "SPP system", will then enable us to determine the NMR structures of membrane proteins without purification and to explore protein dynamics in living cells. Relevance to public health: The technology that will be developed in this study will make a major breakthrough in the structure biology of membrane proteins, which play numerous crucial roles in human health and human diseases. Relevance to public health: The technology that will be developed in this study will make a major breakthrough in the structure biology of membrane proteins, which play numerous crucial roles in human health and human diseases. One of the major challenges in molecular biology during this decade is the determination of three-dimensional structures of membrane proteins, which play various essential roles in living cells and thereby are directly related with human health. The analysis of genomes from bacteria to humans reveals that as much as 30% of the total proteins are, indeed, membrane proteins. However, in spite of the fact that as much as 17,780 three- dimensional structures have been determined, only 0.56% of these structures are of membrane proteins. The major reason for this surprising lack of structural studies on membrane proteins is due to the extreme difficulty of membrane protein purification and their extremely poor yields. To circumvent these problems, I will attempt to establish the technology needed to produce a single membrane protein of interest in living cells without producing any other cellular proteins. This technology, termed the "SPP system", will then enable us to determine the structures of membrane proteins without purification and to explore protein dynamics in the living cells. This will, no doubt, revolutionize the structural biology of membrane proteins and will make a major contribution to our understanding of how membrane proteins function in living cells.